Circulating tumor cells (CTC) are cells that have detached from primary tumors, and entered the blood stream. They have the potential to seed new tumors at distant sites causing propagation and metastases. It is estimated that in a cancer patient, more than 1 million tumor cells per gram of tumor tissue enter the bloodstream every day. This shedding process is discontinuous, and detected CTC are heterogeneous, some destined to never succeed at implantation. Indeed, colonization of distant organs by CTC is an extremely inefficient process, and the vast majority of these cells may either be destroyed in the circulation, or become dormant at distant sites due to the absence of proper growth regulatory niches. However, once metastasis is established, the subsequent seeding of cancer cells may become much more efficient and deadly. Inherent to this is the amplification and change of the CTC pool during the sequential cycles of cancer cell dissemination. While many questions surrounding these events remain unanswered, the accurate detection and characterization of CTC may shed new light on the aggressiveness and metastases potential of the underlying disease. Thus early detection of low counts of CTCs per blood sample is extremely important as it can be used as a valuable indicator for patients and doctors for diagnosis and to block metastases.
CTCs dimensions may range between ˜5-15 μm in diameter and are slightly larger than red blood cells (erythrocytes or RBCs) that are biconcave in shape and ˜8 μm in diameter and 2 μm in height, but no larger than the white blood cells (WBCs or leukocytes) that are spherical in shape and 8-15 μm in diameter. Therefore, the detection and separation of the CTCs is extremely difficult using standard cell separation technologies. The outstanding question is, “How can we detect 1 to 10 CTCs mixed with 5 billion red and white blood cells!?”
There have been several efforts by other researchers to capture and quantify low doses of CTCs in patients' blood but due to technological hurdles it has been very difficult to accurately count and detect CTCs at early stages of cancer development since these cells are rare. The ideal CTC detector system should have the following characteristics: (1) accurately detect and count CTC cells, (2) count the number of CTCs in short period of time (within a few minutes), (3) use peripheral blood sample without requiring any purification or enrichment methods, (4) inexpensive so it can be acquired by several clinical setting, (5) simple enough to be used by primary physician, nurse practitioners and technicians, and (6) portable enough to administer such testing at distant sites similar to point of care testing.
Current Methods for CTC Detection
CTC detection methods can be classified into two types: (1) immunological assays that use monoclonal antibodies, and (2) Polymerase Chain Reaction (PCR) based methods that detect tumor specific antigens. Immunological methods have been widely used for CTC detection. The choice of appropriate markers is a challenge as antigens exclusively expressed by CTC and not shared by other circulating non-tumor or blood cells are scarce. Antibodies specific to epithelial antigens such as cytokeratin, and epithelial cell adhesion molecule (EpCAM) are the most widely used markers for epithelial tumor cell detection. Organ-specific markers, including prostate specific antigen (PSA), carcinoembryogenic antigen (CEA) or HER-2 have also been used. However, they are prone to false-negative/positive results as these markers are not necessarily present in all tumor cells (only up to 30% of cancer cells carry HER-2 in HER-2-positive breast cancer) or are not entirely organ specific. More recently prostate specific membrane antigen (PSMA) based CTC detectors have been created that can detect prostate cancer cells with high efficiency. Several immunofluorescence-based technologies are being used and aim to improve the threshold of detection. Enrichment methods with anti-cytokeratin or combination of anti-cytokeratin and anti-EpCAM antibodies have shown to improve the enrichment process for CTCs that has low EpCAM expression.
Several technologies are available for detection of CTCs in blood. Traditionally, density gradient centrifugation is the method that has been used for isolation of CTCs for microscopy. Heavier components in the blood with higher sedimentation rates are separated from the lighter mononuclear components including tumor cells. These are then transferred to slides and stained for epithelial markers such as
EpCAM to detect CTC. A trained pathologist should examine the slides for CTC in a time consuming process (one to several days for each sample), subject to false positives and/or false negatives depending on the skill of the operator. Moreover density gradient centrifugation has only a recovery rate of 70%. The downfall of using many of these gradient liquids is that whole blood tends to mix with the gradient if not centrifuged immediately; therefore, interrupting total separation.
Isolation of CTCs using polycarbonate filters have been demonstrated in the past. It is inexpensive and a simpler form of enrichment and capture of CTC. The polycarbonate filters have track etching that results in random placement of pores. This results in low density, and often results in fusion of two or more pores together. They have claimed efficiency of capture is 50-60%. In one of the new devices, paralyne C microfilter assembly is used as a modified form of polycarbonate filters for the capture of CTCs with a yield of 90%. In general most polycarbonate filters suffer from the some drawbacks as mentioned above making it prone to produce false positive or negative results.
CellSearch (Veridex) is the first wide spread CTC detector that has been approved by the Food and Drug Administration (FDA). It works for epithelial cancers namely breast, colon and prostate cancers. The system is based on the enumeration of epithelial cells, which are separated from the blood by antibody coated magnetic beads and identified using fluorescently labeled antibodies against cytokeratin and with a fluorescent nuclear stain and fluorescent cytokeratin antibodies. In their original study using the Veridex system, a total of 177 breast cancer patients were enrolled and tested for their CTC counts over a period of two years. Outcomes were assessed according to levels of CTCs at the baseline, before patients started a new treatment for metastatic disease. It was found that patients in a training set with levels of CTC cells equal to or higher than 5 CTCs per 7.5 ml of whole blood, as compared to with fewer than 5 CTC per 7.5 ml, had a shorter median free progression survival and a shorter overall survival. Systems such as CellSearch, however, suffer from several drawbacks. Multiple steps of batch purification and enrichment result in CTC loss. The actual number of CTCs might be much higher to start within each patient group. Secondly, it might be difficult to fish out cells that do not express EpCAM, possibly because the cells have undergone epithelial mesenchymal transformation (EMT), which makes the cells less susceptible to stick to the antibodies as they break free into the blood circulation. EpCAM methods are also not useful for non-epithelial cancers such as sarcomas. Nevertheless, this is the only FDA approved CTC detector currently in the market.
Another technology that uses passive microfluidic sorting of cells in blood is the CTC chip. The CTC chip has 78,000 micro-posts that is etched in silicon. Antibodies such as EpCAM are functionalized on the surface of the micro-posts. Anti-EpCAM provides the specificity for CTC capture from unfractionated blood as it is overexpressed in epithelial cells and is absent in hematologic cells. The CTC chip measured the number of CTCs in peripheral blood of patients with metastatic lung, prostate, pancreatic, breast and colon cancer in 115 of 116 samples with a range of 5-1,281 CTCs per ml and approximately 50% purity. The CTC chip efficiency depends on the velocity of the blood flow and the resulting drag force on the cells, because it influences the duration of the cell-micropost contact and the chance of subsequent attachment. Therefore, the flow rates are kept extremely low in the order of 1.0 ml/hr. With such a small flow rate, the CTC chip essentially takes 6-8 hours of sorting time for one sample of 7.5 ml of patient blood, followed by confocal microscopy. The yield of this CTC capture is less than 20% at 3.0 ml/hr flow rates. The shear forces around micro-post also make the cell circumvent around the posts thereby making this device prone to false negative results.
Therefore there is need for a portable Circulating Tumor Cell (CTC) detector, such as one example of the present invention that can detect less than 10 CTCs per milliliter in a patient's blood. The ideal CTC detector should utilize pure blood as extracted from the patient blood before any purification or additional enrichment steps, which may increase the probability of losing CTCs. Also the ideal CTC detector must be inexpensive, disposable and fast so it can be used in a physician's office and reveal the results in a few minutes. With recent progress in micro and nano fabrication technology, it is now possible to adopt new approaches to capture and count CTCs. Some of these methods are; isolation of CTCs using polycarbonate filters, Parylene micro-filters nanotube biosensor, Ferro Fluid/Magnetic nanoparticles and three dimensional nanostructured array to capture and count CTCs. However, due to challenges associated with each of these methods, the fabrication of a handheld CTC detector with sub 10 CTCs per ml is still not a reality.